Principles of Fluorescence Spectroscopy

178 FREQUENCY-DOMAIN LIFETIME MEASUREMENTS
Figure 5.26. Frequency-domain measurements of the intensity decay
of NADH up to 2-GHz. From [3].
a vasoconstrictor. Vasopressin is a cyclic polypeptide that
contains 9 amino acids including a single tyrosine residue
at position 2. Oxytocin has a similar structure, but has a distinct
physiological activity of stimulating smooth muscle
contraction. Hence there have been many efforts to use the
tyrosine emission to learn about the solution conformation
of these peptide hormones. The frequency-domain data for
vasopressin reveal a complex intensity decay (Figure
5.27). 114 The decay is not even closely approximated by the
single exponential model (dashed). Fitting the data requires
three decay times of 0.17, 0.75, and 1.60 ns. These multiple
decay times could probably not be recovered if the data
were limited to 200 MHz.
MCP PMTs are moderately expensive, and their use in
FD measurements requires special circuits for cross-correlation.
However, the advantages of high-frequency FD data
may become available without the use of MCP PMTs.
Examination of Figures 5.26 and 5.27 indicates that considerable
data can be obtained if the data were available to just
1 GHz. This frequency limit can probably be reached with
the new compact PMTs, which show short transit time
spreads (Table 4.1). A dynode PMT (H5023) has already
been used up to 1 GHz. 115 It seems likely that a compact
PMT such as the R74000 will be useful up to 900 MHz.
Laser diodes with 30-ps pulse widths will provide useful
harmonics up to 40 GHz.
Figure 5.27. Phase and modulation data for the vasopressin tyrosine
fluorescence intensity decay. The dashed line is the best single-exponential
fit, and the solid line is the best three-exponential fit. From
[114].
In the near future gigahertz FD instruments will be
available based on pulsed-laser diodes and compact PMTs.
5.7. ANALYSIS OF FREQUENCY-DOMAIN DATA
Frequency-domain data is often analyzed in terms of the
multi-exponential model. As described in Chapter 4, the
amplitudes (α i ) and decay times (τ i ) are usually strongly
correlated, so that there can be considerable uncertainty in
the values of the recovered parameters. In this section we
describe examples that show correlation between the
parameter values.
5.7.1. Resolution of Two Widely Spaced Lifetimes
The analysis of frequency-domain data can be illustrated
using a mixture of p-terphenyl and indole. This same mixture
was used in Chapter 4 for TCSPC data (Figures 4.46–
4.49). The same 292-nm excitation and 330-nm emission
wavelengths were used for the frequency-domain measurements
as for the time-domain measurements. The decay
times of the individual fluorophores are 0.93 and 3.58 ns for
p-terphenyl and indole, respectively. For this mixture the
decay times are spaced 3.8-fold, making this a moderately